CN1201489A - Alkaline protease, process for production thereof, using method thereof and microorganism producing same - Google Patents
Alkaline protease, process for production thereof, using method thereof and microorganism producing same Download PDFInfo
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- CN1201489A CN1201489A CN96198013A CN96198013A CN1201489A CN 1201489 A CN1201489 A CN 1201489A CN 96198013 A CN96198013 A CN 96198013A CN 96198013 A CN96198013 A CN 96198013A CN 1201489 A CN1201489 A CN 1201489A
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
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- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
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- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38609—Protease or amylase in solid compositions only
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- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C11D3/38618—Protease or amylase in liquid compositions only
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- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention relates to a novel strain of Bacillus sp.(accession number: FERM BP-5736) and a production process and the use of a protease which is obtained by incubating this strain or its variant, shows a high stability in the presence of surfactants, and has the following properties: (1) hydrolyzing proteins and peptides; (2) having an optimum pH value of around 12, when reacted with the use of casein as the substrate at 30 DEG C for 10 minutes; (3) having a stable pH value of from 5 to 11, when incubated with the use of casein as the substrate at 30 DEG C for 24 hours; (4) having an optimum action temperature of around 60 DEG C, when reacted with the use of casein as the substrate at pH 10 for 10 minutes; (5) having a molecular weight of 29,000 +/- 2,000 determined by SDS polyacrylamide gel electrophoresis; (6) having an isoelectric piont of 10.1 +/- 0.5 determined by isoelectric polyacrylamide gel electrphoresis; and (7) having a residual activity of 60% or above in a 500ppm LAS solution after 30 minutes at 40 DEG C or 20% or above after 15 minutes at 55 DEG C.
Description
Technical field
The present invention relates to a kind of new Sumizyme MP.More particularly, the present invention relates to a kind of in tensio-active agent, stablizing and heat-proof Sumizyme MP, its production method, its microorganism of using and producing Sumizyme MP in stain remover.
Background technology
In recent years, environmental pollution had become a social concern.In order to regulate the use of phosphoric acid salt, enzyme is mixed with stain remover to improve detergency as the stain remover composition.The stain remover that contains enzyme (for example proteolytic enzyme, amylase, cellulase and lipase) has been arranged in the market.Particularly, the proteolytic enzyme organic stain on the clothes of can degrading, organic stain of 10-40% is caused by protein, and this stain can not be washed off by conventional stain remover.Proteolytic enzyme has become a kind of neccessary composition that improves detergency.
As the proteolytic enzyme that uses in the stain remover, have much to derive from and in the alkaline pH scope, have active microorganism, and used proteolytic enzyme, as Kazusase (Showa Denko K.K.), Savinase (Novo), Maxacal (Gist), Alcalase (Novo) and Biosam (ShowaDenko K.K.).Along with the widespread use of automatic dishwasher, demand can be used in the enzyme of automatic dishwasher now.Proteolytic enzyme is effective to protein stains such as the yolk on the plate, milk preparations.Yet, in general during high temperature in the aqueous solution inactivation of enzyme accelerate.Therefore, the enzyme that need have advantage aspect stable.Although present Esperase (Novo) etc. has been used for this purpose, these enzymes do not make us the active and stable of satisfaction fully.The most stable proteolytic enzyme of knowing at present in tensio-active agent is to produce by Bacillus strain SD 521 (number of patent applications 191781 in 1991).Yet, need a kind of more stable proteolytic enzyme.
Therefore, the purpose of this invention is to provide a kind of Sumizyme MP more stable in tensio-active agent, the method for producing this Sumizyme MP, this proteolytic enzyme in detergent compositions application and produce the microorganism of this proteolytic enzyme.
Invention is described
The result that we make great efforts to study has found to have fabulous stability by the protease A PI-26 that bacillus unknown species SD 114 (bacterial strain of a kind of bacillus) produces, and is suitable for using in stain remover, and has finished the present invention.
Therefore, the invention provides following new Sumizyme MP, its microorganism of production method, purposes and this proteolytic enzyme of generation.
1) satisfy the Sumizyme MP of at least one condition of following (a)-(c) defined:
(a) in surfactant soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA, 500ppm LAS), place that remaining activity surpasses 60% after 30 minutes under 40 ℃
(b) in buffer agent solution (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA), place that remaining activity surpasses 40% after 15 minutes under 55 ℃
(c) in surfactant soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA, 500ppm LAS), place that remaining activity surpasses 20% after 15 minutes under 55 ℃
2) the above the 1st) in the regulation the Sumizyme MP with following properties:
(1) effect
This proteolytic enzyme can protein hydrolysate and peptide.
(2) optimal pH
When this proteolytic enzyme under 30 ℃, be that the best pH of substrate reactions in the time of 10 minutes is approximately 12 with the casein.
(3) stable pH range
With this proteolytic enzyme incubation in the time of 24 hours, it is stable in the pH of 5-11 scope under 30 ℃.
(4) optimum temps
When this proteolytic enzyme during, be that the optimum temps of substrate reactions in the time of 10 minutes is approximately 60 ℃ with the casein at pH10.
(5) molecular weight
The molecular weight that records by the SDS-polyacrylamide gel electrophoresis is 29,000 ± 2,000.
(6) iso-electric point
The iso-electric point that records by the isoelectrofocusing polyacrylamide gel electrophoresis is 10.1 ± 0.5.
3) the above the 1st) or the 2nd) in the regulation the Sumizyme MP by the microorganisms that belongs to bacillus.
4) the above the 3rd) in the regulation the Sumizyme MP by the microorganisms that belongs to bacillus, wherein said microorganism is bacillus unknown species SD114 (preserving number FERM BP-5736).
5) with the above the 4th) in the proteolytic enzyme of regulation have the Sumizyme MP of immune cross-reactivity, and can satisfy at least one condition of following (a)-(c) defined:
(a) in surfactant soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA, 500ppm LAS), place that remaining activity surpasses 60% after 30 minutes under 40 ℃.
(b) in buffer soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA), place that remaining activity surpasses 40% after 15 minutes under 55 ℃.
(c) in surfactant soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA, 500ppm LAS), place that remaining activity surpasses 20% after 15 minutes under 55 ℃.
6) a kind of method of producing Sumizyme MP is characterized in that from producing the above the 1st)-5) obtain said proteolytic enzyme the microorganism that belongs to bacillus of the proteolytic enzyme of defined or the culture of its mutation.
7) a kind of production the above the 6th) method of the Sumizyme MP of a defined, said proteolytic enzyme is by the microorganisms that belongs to bacillus, and this microorganism is bacillus unknown species SD114 (FERM BP-5736).
8) bacillus unknown species SD 114 (FERM BP-5736) and its mutant.
9) composition of said stain remover is comprising the above the 1st)-5) proteolytic enzyme of defined.
10) comprise the above the 1st)-5) stain remover of the proteolytic enzyme of defined.
11) be used for the stain remover of automatic dishwasher, wherein comprise the above the 1st)-5) proteolytic enzyme of a defined.
12) produce peptide or amino acid whose method, it is characterized in that making the above the 1st)-5) proteolytic enzyme and the protein or the reactive polypeptide of arbitrary defined of item.
The letter of accompanying drawing is more described
[Fig. 1] is the figure of the optimal pH scope of description enzyme of the present invention.
[Fig. 2] is the figure of the stable pH range of description enzyme of the present invention.
[Fig. 3] is the figure of the optimum temperature range of description enzyme of the present invention.
[Fig. 4] is for describing the figure of enzyme of the present invention and the stability of known enzyme under 55 ℃.
[Fig. 5] is for describing the figure of the stability of known enzyme under 55 ℃ in enzyme of the present invention and the commercially available stain remover (Ultra Ariel).
[Fig. 6] is for describing the figure of the stability of known enzyme under 55 ℃ in enzyme of the present invention and the commercially available stain remover (Super Cheer).
[Fig. 7] is for describing the figure of the stability of known enzyme under 40 ℃ in enzyme of the present invention and the commercially available stain remover (Tide Grease Releasing).
The microorganism that openly produces in detail enzyme of invention
The bacillus unknown species bacterial strain SD 114 of one of microorganism of generation enzyme of the present invention is a kind of and strong bacillus (Bacillus firmus) closely-related bacterial strains on mycology. Yet this bacterial strain is at the known bacterial strain that is different from significantly other aspect some characteristics, and it is considered to a new bacterial strain. Bacteriology characteristic:
Bacillus unknown species bacterial strain SD 114 related to the present invention has following bacteriology characteristic:
(a) form: bacillus
(b) Gram’s staining: the positive
(c) generation of spore: the positive
(d) shape of spore: ellipse
(e) mobility: the positive
(f) to the reaction of oxygen: aerobic bacteria
(g) catalatic generation: the positive
(h) under anaerobic growth: feminine gender
(i) vogesprokauer reaction (VP) reaction: feminine gender
(i) pH:6.2 of VP broth culture
(k) generation of acid: glucose: feminine gender
Arabinose: feminine gender
Wood sugar: feminine gender
Mannitol: feminine gender
(i) by the glucose aerogenesis: feminine gender
(m) liquefaction of gelatin: the positive
(n) degraded of starch: the positive
(o) utilization of citrate: feminine gender
(p) utilization of propionate: feminine gender
(q) phenylalanine deamination: feminine gender
(r) egg yellow reaction: feminine gender
(s) reduction of nitrate: feminine gender
(t) growth under pH6.8: the positive
(u) growth under pH5.7: the positive
(v) growth in 5% sodium chloride: the positive
(w) growth in 7% sodium chloride: the positive
(x) growth under 10 ℃: the positive
(y) growth under 30 ℃: the positive
(z) growth under 55 ℃: the positive
(aa) the GC content of microbial DNA (% by mole): 45%
With reference to the systematic bacteriology handbook (Vol.2, William and Wilkins, 1986) of Bergey, system features of said bacterium with above-mentioned bacterial characteristics and other bacterial strain are compared as follows:
The bacillus unknown species bacterial strain SD 114 that the present invention relates to does not show the feature identical with strong genus bacillus aspect acid and the 50 ℃ of growths being produced by sugar.Yet known bacterial strain SD 521 is produced acid, is made the nitrate deoxidation by glucose, can not grow when pH6.8, can not be 50 ℃ of growths.NCIB 10309 produces acid by glucose, pectinose and mannitol.PB 92 produces acid by glucose and wood sugar, and can utilize propionic salt.Can find out significantly that from above bacterial characteristics bacterial strain SD 114 is a kind of thermophilic bacterias, be and the closely-related bacillus unknown species of strong genus bacillus, but this bacterial strain is different with other known bacterial strain.Therefore this bacterial strain is considered to a kind of new bacterial strain.This bacterial strain is deposited in bio-science and human technology Consiglio Nazionale Delle Ricerche (IT) T, Piazzale Aido Moro-00185 Rome, Italy (industrial science and technical body (address: 1-3 in October 2 nineteen ninety-five with FERM P-15214, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan)), be transformed into international preservation thing according to budapest treaty with bacterial strain SD 114 (FERM BP-5736) and on October 30th, 1996.
In addition, the mutant (obtaining by spontaneous or induced mutation) that belongs to the proteolytic enzyme improvements in productivity of said bacterial strain SD 114 can be used as the bacterium that produces proteolytic enzyme of the present invention.In order to prepare these mutant, can use conventional method, after for example original strain being induced artificial mutation, culture is inoculated in the nutrient agar that contains skimming milk etc. by uv-radiation or mutagenic compound (as N-methyl-N '-nitro-N-nitrosoguanidine (NTG)).The mutant that selection has excellent productivity from the bacterium colony that forms bigger clear endless belt in periphery of bacterial colonies.And can improve productivity by in proper host cell, utilizing the gene amplification phenomenon, use the said host cell of gene transformation with suitable self-replacation DNA link coupled proteolytic enzyme of the present invention.Produce the method for proteolytic enzyme
Any substratum that can make the microorganism growth that produces proteolytic enzyme of the present invention and produce said proteolytic enzyme may be used to produce proteolytic enzyme of the present invention.
For example, can use glucose, maltose, sucrose, Zulkovsky starch etc., use organic nitrogen compound (as soybean cake, chaff cake and corn immersion liquid) as nitrogenous source as carbon source.To add mineral substance in addition, as phosphoric acid salt, sylvite and magnesium salts.In the present invention, culture is cultivated under aerobic condition, for example can be used aerated culture or wave and culture.Although this bacterial strain is stable under 20-40 ℃ temperature, the culture temperature that requires to use is 30-37 ℃.The initial pH of ideal is 9-10, and pH in the training period is 8.5-10.Incubation time is 16-60 hour, and finishing to cultivate when obtaining maximum protease activity is ideal.Can carry out purifying to the culture that obtains by above process according to the separation and the purification process of routine.By salt precipitation protein, solvent fractionation method, spray drying process, lyophilization or the like, soluble salt or hydrophilic organic solvent are joined in the supernatant liquor of gained or the filtrate (said supernatant liquor and filtrate are to obtain by solid residue centrifugal or that remove by filter somatocyte or substratum), obtained proteolytic enzyme of the present invention.And the purification process (as ion exchange chromatography and gel permeation chromatography) that can be used in combination other is further purified said proteolytic enzyme.We are the proteolytic enzyme called after API-26 of the present invention that is obtained by the way.Measure the activity of API-26 by following method.The mensuration of enzymic activity
The enzyme solution that 50 μ l are suitably diluted joins in the 500 μ l 50mM Atkins-Pantin borate buffer solutions (pH10), and at 30 ℃ of following preincubation 3-5 minutes.The 2% Han Masitanshi casein solution (pH10) of 500 μ l is joined in this solution, and after 10 minutes, trichoroacetic acid(TCA) (TCA) solution (being adjusted to the 0.032M TCA solution of pH4 with acetate buffer) that adds 2ml makes reaction terminating.Place more than 10 minutes down at 30 ℃, use No. 2 filter paper (Toyo Roshi) to filter then.The phenol reagent of 6 times of the 0.4M yellow soda ash of 5ml and 1ml dilute with waters is joined in the 1ml filtrate.Descended painted 20 minutes at 30 ℃, be determined at the absorbancy under the 660nm then.Katal is a unit of describing protease activity.1 katal be defined as pH10 with 30 ℃ under be the degraded product that produces of substrate protease activity identical under 660nm with the casein with the absorbancy of 1 mole of tyrosine.The characteristic of proteolytic enzyme
The physics and the chemical property of the proteolytic enzyme that the present invention obtained are as follows:
(1) effect and substrate specificity
This proteasome degradation protein or peptide (as casein, oxyphorase, albumin, meat protein, fish protein and soy-protein).
(2) optimal pH
Determining of pH value:
With Britton-Robinson wide region damping fluid as buffered soln.In order to determine optimum pH, the enzyme of about 20nkatal/ml is joined each contain in the 1% caseic pH damping fluid.Measure enzymic activity at 30 ℃ of following incubations after 10 minutes.Enzymic activity such as table 1 and shown in Figure 1, wherein the activity under pH11.7 is considered to 100.The optimum pH of finding out this enzyme from these results is approximately 12.
(3) stable pH range
Determining of stable pH range:
The concentration of this enzyme with about 20nkatal/ml is joined in each pH buffered soln.Measure enzymic activity at 30 ℃ of following incubations after 10 minutes.Table 2 and Fig. 2 have described remaining activity, and wherein the activity before incubation is considered to 100.Stable pH value scope at 30 ℃ of following these enzymes is approximately 5-11 as can be seen from these results.
Table 1
| ????pH????7.1??7.9??9.0??9.9??10.?11.?11.?12.1 ???????????????????????????????6???2???7 |
| Active 19 35 46 64 79 86 100 94 |
Table 2
| ????pH????4???5???6???7???8???9???10???11???12 |
| Active 0 100 100 100 100 100 100 100 0 |
(4) optimum temperuture
Determining of optimum temperuture:
After the Atkins-Pantin borate buffer solution (pH10) that under each temperature 25mM is contained 2mM ethylenediamine tetraacetic acid (EDTA) (EDTA) carries out preincubation, add said enzyme and under each temperature the reaction 10 minutes.Under 30 ℃ this solution incubation was measured enzymic activity after 24 hours.Table 2 and Fig. 2 have described relative reactivity, and wherein the activity under 60 ℃ is considered to 100.The optimum temperuture of this enzyme is approximately 60 ℃ as can be seen from these results.
Table 3
| Temperature (℃) | ??30 | ??40 | ????50 | ??60 | ??70 |
| Active | ??13 | ??25 | ????49 | ?100 | ??35 |
(5) influence of inhibition
Studied of the influence of various inhibitions according to following condition and method to said enzyme:
Preparation contains 50mM borate buffer solution (pH10) solution of about 20nkatal/ml enzyme.With EDTA, mercury phenylformic acid (PCMV) or tolylsulfonyl fluorine (PMSF) are joined in this solution with the concentration shown in the table 4.30 ℃ of following incubations were measured enzymic activity after 30 minutes.The result is as shown in table 4, and the relative reactivity of enzyme is not considered to 100 when wherein having inhibition.As shown in table 4, PMSF suppressed this enzyme in ten minutes consumingly, and therefore said enzyme is a serine protease.
Table 4
| Inhibition | Concentration | Active |
| There is not inhibition | ????100 | |
| PCMV | ????1mM | ????100 |
| PMSF | ????10mM | ????0.7 |
| EDTA | ????5mM | ????99 |
(6) molecular weight
The molecular weight of this enzyme that records by the SDS-polyacrylamide gel electrophoresis is 29,000 ± 2,000.
(7) iso-electric point
The iso-electric point that records by the isoelectrofocusing polyacrylamide gel electrophoresis is 10.1 ± 0.5.
(8) stability
Said endonuclease capable satisfies one of following at least condition:
(a) in surfactant soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA, 500ppm linear alkyl benzene sulfonate sodium (LAS)), place that remaining activity surpasses 60% after 30 minutes under 40 ℃.
(b) in buffered soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA), place that remaining activity surpasses 40% after 15 minutes under 55 ℃.
(c) in surfactant soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA, 500ppm LAS), place that remaining activity surpasses 20% after 15 minutes under 55 ℃.Cross reactivity with anti-API-26 body
Proteolytic enzyme of the present invention preferably shows the cross reaction with anti-API-26 body under following condition determination.
The condition determination of cross reaction: the API-26 antigen of preparation 2mg/ml, and the Freund's complete adjuvant of itself and equivalent mixed to form water-in-oil emulsion completely.This emulsion of 1ml was administered on 2 does at 0 day, 21 days, 42 days and 63 days.In the time of 70 days, adopt avascularization to collect all blood, and preparation contain the serum sample of antibody.
Use Ouchterlony technology (Acta.Med.Scan.133:76-79,1950) assessment cross reaction, that is: fill medium pore with API-26 or other proteolytic enzyme of 10 μ l with the concentration of 1mg/ml, and fill hole on every side with 10 μ l antiserum(antisera)s (with 2n (n=1,2,3,4,5,6,7) dilution proportion).Flat board is placed in the humidity culturing room, and under 37 ℃, is incubated overnight, up to seeing the precipitin line.If the precipitin line is when forming with 4 times of the Cmin of the precipitin line that forms API-26 or greater concn, just can draw said enzyme and anti-API-26 body has cross reactivity.The application of proteolytic enzyme
The protease treatment protein or the peptide of the application of the invention can produce peptide or amino acid.Can be with this proteolytic enzyme editing objective material by using protease treatment to comprise the target substance of protein or peptide.In the using method of routine, proteolytic enzyme of the present invention can share with the proteolytic enzyme of routine.Even under the severe condition of conventional enzyme deactivation, proteolytic enzyme of the present invention can also use up to this proteolytic enzyme and be activated.For example, not only can when 50ppm-500ppm LAS exists, use said proteolytic enzyme, and also can use above in the presence of the LAS of 500ppm.Yet less than 3, it is preferred that 000ppm uses this proteolytic enzyme down, is preferred use this proteolytic enzyme less than 300ppm.The said proteolytic enzyme of use under 48 ℃ the high temperature can surpassed.Yet using this proteolytic enzyme down less than 75 ℃ is preferred, is preferred using this proteolytic enzyme down less than 70 ℃.Purposes
In various commercially available detergent solutions or tensio-active agent, Sumizyme MP of the present invention is more stable than conventional Sumizyme MP.Because this enzyme also has thermostability, so with warm water washing clothes or plate the time, it is the degrade proteins stain effectively, so can increase detergency by in stain remover, adding this proteolytic enzyme.
In addition, this proteolytic enzyme can be used for feed processing, food-processing (fish oil processing, meat processing etc.), fiber process, wool processing, leather processing, the washing of contact lens, the washing of pipeline.In addition, this proteolytic enzyme can mix use with body lotion and trichogen.Detergent compositions
The Sumizyme MP blended detergent compositions that the invention provides and have above-mentioned specified characteristic.Though the quantity of unqualified and of the present invention detergent compositions blended Sumizyme MP will be equivalent to 10-1, it is suitable that the enzyme of the detergent solution amount of 000nkatal/l mixes.If the amount that adds is too little, just improve detergency unsatisfactorily.On the contrary, if the amount that adds is too many, can not resembles and improve detergency desired, and consider it also is worthless from economic aspect.
Sumizyme MP of the present invention can mix with known detergent compositions and no any change on forming.Detergent compositions of the present invention does not need special composition.The typical example of this detergent compositions is: anti-sludging agent, enzyme, SYNTHETIC OPTICAL WHITNER, fluorescent dye, anti-caking agent and antioxidant (weight according to detergent compositions is decided) with the alkaline reagents of auxiliary agent, 1-50% (weight ratio) of tensio-active agent, the 0-50% (weight ratio) of the detergent compositions of forming that one or more are made of following material: 10-50% (weight ratio) or mineral substance, 0.1-5% (weight ratio).
Can use the following surfactant that contains usually in the detergent compositions: soap; Line style or branch's alkyl or alkenyl sulfuric ester; The amidosulphuric acid ester; The analiphatic sulphur acid compound, as have the alkyl of linearity or branch's alkyl or kiki alkenyl group (wherein having added one or more oxyethane, propylene oxide and butylene oxide ring) or alkenyl ether sulfuric ester, alkyl sulfonic ester, sulfamate, dialkyl sulfosuccinate succinate, aliphatic sulphonic acid ester (as the sulphonate of alpha-olefin, vinylidene alkene and internal olefin); Aromatic sulfonic acid ester is as line style or ramose benzene sulfonamide acid esters, have the alkyl or the alkenyl ether carboxylicesters of line style or branch's alkyl or kiki alkenyl group (wherein having added one or more oxyethane, propylene oxide and butylene oxide ring); Alpha-sulfo-fatty acid or ester; The tensio-active agent of amino acid pattern; Alkyl or alkenyl acid phosphoric acid ester; Phosphate ester surfactants (as alkyl or alkenyl phosphoric acid ester); The amphoterics of sulfonic acid type; The betaine type amphoteric surfac-tant; The alkyl or alkene ether or the alcohol that have line style or branched alkyl groups (wherein having added one or more oxyethane, propylene oxide and butylene oxide ring); The polyoxyethylene alkyl phenylate that has line style or branched alkyl groups (wherein having added one or more oxyethane, propylene oxide and butylene oxide ring); Higher fatty acid alkanolamide or their alkylene oxide addition compound; Sucrose fatty ester; Glycerine fatty acid one ester; Alkyl or alkenyl amine oxide compound; Tetraalkylammonium salt type cats product; Or the like.The counter ion of anion surfactant are sodium ion or potassium ion preferably.Said stain remover can contain one or more these surfactant mixtures.
Following mineral compound can be used as washing assistant and basifier or inorganic electrolyte.What can be used as alkaline metal salt has: phosphoric acid salt (as orthophosphoric acid salt, pyrophosphate salt, tri-polyphosphate, metaphosphate, hexametaphosphate, phytate or the like); Phosphonate (as ethane-1,1-di 2 ethylhexyl phosphonic acid, ethane-1,1,2-tri methylene phosphonic acid, ethane-1-hydroxyl-1,1-di 2 ethylhexyl phosphonic acid and their derivative, ethane hydroxyl-1,1,2-tri methylene phosphonic acid, ethane-1,2-dicarboxyl-1,2-di 2 ethylhexyl phosphonic acid, methane hydroxyethylidene diphosphonic acid or the like); Phosphono-carboxylic acids salt (as 2-phosphinylidyne butane-1,2-dicarboxylic acid, 1-phosphinylidyne butane-2,3,4-three dicarboxylic acid, α-methylphosphine acyl group succsinic acid or the like); Amino acid salts (as aspartic acid, L-glutamic acid or the like); Amino polyacetic acid salt (as nitrilotriacetic acid(NTA), edetate, diethylentriamine pentacetate or the like); Polymer electrolyte (as polyacrylic acid, polymethylene succinate, the multipolymer that gathers maleic acid, anhydrous maleic acid, carboxymethyl cellulose salt or the like); Non-dissociated polymkeric substance (as polyoxyethylene glycol, polyvinyl alcohol or the like); Carboxymethylation compound (as diethyl alkyd, hydroxyl diethyl alkyd, carboxymethyl oxysuccinic acid, citric acid, lactic acid, tartrate, sucrose, lactose or the like); Organic acid salt (as the carboxymethylation compound of the carboxymethylation compound of tetramethylolmethane, glyconic acid, benzene polycarboxylic acid, oxalic acid, oxysuccinic acid, hydroxyl two Succinic Acid, glyconic acid or the like); Silico-aluminate (as zeolite or the like); Inorganic salt (as carbonate, sesquicarbonate, vitriol, bisilicate or the like) as alkaline metal salt; As the starch of organic compound, urea or the like; As the sodium-chlor of mineral compound, bentonite or the like; With the trolamine as organic basifier, diethanolamine, Monoethanolamine MEA BASF, tri-isopropanolamine or the like.
As mentioned above, detergent compositions of the present invention contains one or more tensio-active agents, Sumizyme MP of the present invention and basifier or inorganic electrolyte as main component.In addition, if desired, it can contain amphoterics, SYNTHETIC OPTICAL WHITNER (as SPC-D, Sodium peroxoborate etc.), bleach activator, staining agent, washing assistant, anti-redeposition agent (as polyoxyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose etc.), anti-caking agent, antioxidant, sanitas, brightener, agent and other enzyme (as lipase etc.) spume.
Can use any method that said enzyme and detergent compositions of the present invention are mixed.Yet, for to the safety and health of the workman in stain remover user or the stain remover factory harm consider (during the processing of stain remover, producing dust), preferably the fine powder of enzyme is not mixed.Therefore, preferably enzyme is mixed with liquid or dustless form.Method that can be by any routine (as rolling granulate, be squeezed into granular, become granular and become granular through centrifugal fluidized bed through fluidized-bed) with the enzyme moulding.Be used for the shape that the shape with detergent compositions blended enzyme of the present invention is not limited to produce according to the method described above.Because Sumizyme MP of the present invention is very stable, can (for example greater than 50 ℃) carry out the preparation of enzyme under than the higher temperature of conventional preparation method.
The specific form of detergent compositions of the present invention comprises the stain remover of the washing clothes that contains Sumizyme MP of the present invention and is used for the stain remover etc. of automatic dishwasher.Realize best conditions of the present invention
Be to specify example of the present invention below, but the present invention is not only limited to following example.
Used specified in these embodiments commercially available stain remover.Before using them, extract the enzyme granulate that is included in the said stain remover by methods such as sortings and prepare the stain remover that does not contain enzyme.
Embodiment 1: the cultivation of bacillus unknown species bacterial strain SD 114
Under 35 ℃ bacterial strain SD 114 concussion in 300 milliliters of substratum that contain 1% casein, 1% meat soup and peptone more than 1% was cultivated 66 hours,, in culture, produce the said enzyme of justacrine then with the pH regulator to 7.5 of yellow soda ash with substratum.Under 4 ℃ with centrifugal this culture of 1000 G so that obtain supernatant liquor.Enzymic activity in the supernatant liquor is approximately 50nkatal/ml.
Embodiment 2: the purifying of enzyme API-26
The supernatant liquor of the culture that will obtain in embodiment 1 concentrates with ultra-filtration membrane then with removing the membrane filtration that degerms.With the ammonium sulfate that 30-60% is saturated this spissated solution is saltoutd.(pH7.5 comprises 1mM CaCl at 25mM tris-HCl damping fluid with the resolution of precipitate that obtains
2) in, and dialyse with this damping fluid.Under pH7.5, this solution is joined in the CM-cellulofine C-500 post (Seikagaku company) then, use 0-1M to contain 1mM CaCl
2KCl by the said enzyme of concentration gradient method wash-out.Demonstrate about 8 times increase with the ratio work of comparing the enzyme component before the ion exchange chromatography.It is a single band that this component is carried out this enzyme of SDS-polyacrylamide gel electrophoresis analysis revealed.
Embodiment 3: temperature stability
Enzyme API-26 of the present invention, subtilisin 10309 (by the enzyme of bacterial strain NCIB 10309 generations), PB 92 (by the enzyme of Bacillus strain PB 92 generations) and SD 521 (by the enzyme of Bacillus strain SD 521 generations) are joined 50mM Atkin-Pantin borate buffer solution (pH10, comprise 0.1mM EDTA) in, the solution that activity is approximately 20 nkatal/ml obtained.At 55 ℃ of these solution of following incubation, regularly collect sample, and measure remaining activity down at 30 ℃.Table 5 is the remaining activity of each time point, is 100 in the activity that does not have to measure under the heat treated condition wherein.Method according to No. 191781 interim publication defineds of No. 8401 patent publications in 1976, No. 125407 interim publications in 1976 and 1991 prepares subtilisin 10309, PB 92 and SD 521 respectively.As table 5 and shown in Figure 4, API-26 has fabulous temperature stability.
Table 5. temperature stability (55 ℃)
| Time (minute) 0 7.5 15 22.5 30 |
| API-26 100 70 43 25 30 subtilisins 10,309 100 4333 PB 92 100 5343 SD 521 100 20 13 10 8 |
Embodiment 4: tensio-active agent stability
We have studied the stability of this enzyme to tensio-active agent.Measuring method:
LAS is dissolved in the 50mM Atkin-Pantin borate buffer solution (pH10 comprises 0.1mMEDTA), obtains the LAS solution of 500ppm.(API-26, subtilisin 10309, PB 92 and SD 521) joins in this solution with enzyme, obtains the solution that enzymic activity is approximately 20nkatal/ml, at 40 ℃ and 55 ℃ of these solution of following incubation.Regularly detect remaining activity down at 30 ℃.Table 6 (40 ℃) and table 7 (55 ℃) are the remaining activity at each time point, and enzymic activity is 100 when wherein beginning.Shown in these two tables, compare with conventional enzyme, API-26 has fabulous stability in tensio-active agent.
The stability (40 ℃) of table 6. in tensio-active agent
| Time (minute) 0 7.5 15 22.5 30 |
| API-26 100 100 100 99 97 |
The stability (55 ℃) of table 7. in tensio-active agent
| Time (minute) 00 7.5 15 22.5 30 |
| API-26 100 65 40 20 15 subtilopeptidase As 100 0000 10309 PB 92 100 0000 SD 521 100 3000 |
Embodiment 5: the stability of enzyme API-26 in detergent solution
We have studied enzyme of the present invention at commercially available stain remover UltraAriel (P﹠amp according to following condition and method; G), Super Cheer (P﹠amp; G) and Tide Grease Releasing (P﹠amp; G) stability in the solution.(pH10 comprises 1, the Ca of 000ppm to 50mM Atkin-Pantin borate buffer solution
2+With 1, the detergent solution of 000ppm) middle enzyme API-26 and other enzyme, subtilisin 10309, PB 92 and the SD 521 of adding, obtain the solution that enzymic activity is about 20nkatal/ml, then at 55 ℃ or 40 ℃ of these solution of following incubation.30 ℃ are regularly detected remaining activity down.Table 8-10 and Fig. 5-7 are the remaining activity of each time point, and activity is 100 when wherein beginning.Shown in these tables and figure, compare with other enzyme, API-26 has very remarkable stability in stain remover.
Table 8.Ultra Ariel (55 ℃)
| Time (minute) 0 7.5 15 22.5 30 |
| API-26 100 90 82 75 65 subtilopeptidase As 100 75 48 35 22 10309 PB 92 100 73 46 33 21 SD 521 100 81 53 44 31 |
Table 9.Super Cheer (55 ℃)
| Time (minute) 0 7.5 15 22.5 30 |
| API-26 100 90 80 71 63 subtilopeptidase As 100 25 753 10309 PB 92 100 26 854 SD 521 100 41 25 10 9 |
Table 10.Tide Grease Releasing (40 ℃)
| Time (minute) 0 7.5 15 22.5 30 |
| API-26 100 98 92 84 81 subtilopeptidase As 100 65 40 30 21 10309 PB 92 100 66 41 28 20 SD 521 100 72 66 52 39 |
Embodiment 6: comprise the detergency test of the stain remover of enzyme API-26
Handle the supernatant liquor of the culture that obtains in embodiment 1 identical mode with removing the film that degerms,, prepare the crude preparation by using of this enzyme then by spraying drying with the supernatant liquor of the concentrated gained of ultra-filtration membrane.The raw product enzyme of gained is joined Tide (P﹠amp; G) in (a kind of commercially available liquid detergents), and measure detergency.The concentration of said raw product enzyme with 100nkatal/ml is joined among the Tide.40 ℃ of 4 weeks of stain remover incubation that will contain the enzyme of preparation down.2 stain removers that contain enzyme that restrain 4 weeks of incubation are joined 1 liter contain 40ppm calcium ion (Ca
2+) water in, the washing dirty linen is measured brightness, so as by under definite detergency that establishes an equation.10 piece sizes are that the EMPA-116 of 5cm * 5cm is used as dirty linen.
Detergency (%)=(brightness of the brightness-dirty linen of washing back clothing)/(brightness of the brightness-dirty linen of clean clothing) * 100
Also detect detergency in contrast with SD521.Gained the results are shown in table 11.From these results as can be seen, the adding of API-26 has increased the validity of liquid detergents.
Table 11
| Enzyme detergency (%) |
| API-26 62 subtilopeptidase As 55 10309 PB92 54 SD521 58 do not contain enzyme 52 |
Embodiment 7: contain the detergency test that enzyme API-26 is used for the stain remover of automatic dishwasher
The enzyme crude preparation by using that we use embodiment 6 to describe has been checked its detergency in automatic dishwasher.
Use commercially available stain remover (Hi-wash S (NCC)) preparation to be used for the stain remover that contains said enzyme of automatic dishwasher, said commercially available stain remover contains polyethyleneimine: alkyl oxide, SPC-D, carbonate, vitriol and organic acid salt, and its weight ratio with 20: 1 is mixed with the crude preparation by using of API-26 enzyme.The stain remover of gained is used for washing with 0.21% concentration, and measures detergency.Wash conditions
Machine: by the automatic dishwasher NP-600 of Matsushita power industry company limited assembling, wherein detergent solution is injected from the nozzle of rotation, wash the plate in the detergent solution that is placed on rotation.
Wash temperature: temperature slowly rises to 55 ℃ by 5 ℃
Water: hardness is 3.5 ° of DH
Enzyme concn: 0.01%
The amount of the recirculated water that is used to wash: 2.5 liters
Dirty plate: with 1 gram yolk two diameters of making dirty is the ceramic disc of 25cm, and it is air-dry to spend the night.The evaluation of detergency
After the washing, by take a picture to measure use the area (PI) of the violet region that the amide-Schnitz solution reaction produces on plate.Calculate clean rate according to the planimeter that establishes an equation down by the dirty zone of measuring.
Clean rate (%)={ (P
0-P
1)/P
0* 100}, P wherein
0It is the area of plate.
Table 12 demonstrates the clean rate of calculating.This test also with SD521 in contrast.These results show that the stain remover that contains enzyme API-26 has very strong decontamination activity, this be because itself in addition also be 100% for the clean rate that resembles the obstinate stain the used yolk in this test.Should consider that this enzyme goes far towards to improve the detergency of the stain remover that is used for automatic dishwasher.
Table 12
| Clean rate (%) |
| API-26 100 SD521 98 do not add |
The possibility of industrial application
Alkali protease of the present invention is very stable in commercially available various detergents or surfactant, and comparing it also is stable with existing alkali protease for heat. Because this enzyme is degrade proteins effectively in the washing process of clothing and bowl, so enzyme mixes and can increase detergency therewith.
With this enzyme-treated protein or peptide, it may be economical producing other peptide or amino acid whose process, because this enzyme is stable.
Bacillus unknown species bacterial strain SD 114 of the present invention and mutant thereof are the mesophiles that is easy to process, and its production at alkali protease of the present invention is useful.
Production method by protease of the present invention can be produced alkali protease of the present invention effectively, belongs to microorganism or its mutant of bacillus comprising cultivation.
Claims (12)
1. satisfy the Sumizyme MP of at least one condition of following (a)-(c) defined:
(a) in surfactant soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA, 500ppm LAS), place that remaining activity surpasses 60% after 30 minutes under 40 ℃;
(b) in buffer agent solution (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA), place that remaining activity surpasses 40% after 15 minutes under 55 ℃;
(c) in surfactant soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA, 500ppm LAS), place that remaining activity surpasses 20% after 15 minutes under 55 ℃.
2. the Sumizyme MP with following properties of claim 1 defined:
(1) effect
This proteolytic enzyme energy protein hydrolysate and peptide;
(2) optimal pH
When this proteolytic enzyme under 30 ℃, be that the best pH of substrate reactions in the time of 10 minutes is approximately 12 with the casein;
(3) stable pH range
With this proteolytic enzyme incubation in the time of 24 hours, it is stable in the pH of 5-11 scope under 30 ℃;
(4) optimum temperuture
When this proteolytic enzyme during, be that the optimum temps of substrate reactions in the time of 10 minutes is approximately 60 ℃ with the casein at pH10;
(5) molecular weight
The molecular weight that records by the SDS-polyacrylamide gel electrophoresis is 29,000+2,000;
(6) iso-electric point
The iso-electric point that records by the isoelectrofocusing polyacrylamide gel electrophoresis is 10.1 ± 0.5.
3. the Sumizyme MP by the microorganisms that belongs to bacillus (Bacillus) of claim 1 or claim 2 defined.
4. the Sumizyme MP by the microorganisms that belongs to bacillus of claim 3 defined, wherein said microorganism is bacillus unknown species SD114 (preserving number FERM BP-5736).
With claim 4 in the proteolytic enzyme of regulation have the Sumizyme MP of cross reactivity, and can satisfy at least one condition of following (a)-(c) defined:
(a) in surfactant soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA, 500ppm LAS), place that remaining activity surpasses 60% after 30 minutes under 40 ℃;
(b) in buffer agent solution (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA), place that remaining activity surpasses 40% after 15 minutes under 55 ℃;
(c) in surfactant soln (50mM Atkins-Pantin borate buffer solution (pH10), 0.1mM EDTA, 500ppm LAS), place that remaining activity surpasses 20% after 15 minutes under 55 ℃.
6. method of producing Sumizyme MP is characterized in that obtaining said proteolytic enzyme from the culture of the microorganism that belongs to bacillus of the proteolytic enzyme that produces claim 1-5 defined or its mutation.
7. method of producing the Sumizyme MP of claim 6 defined, said proteolytic enzyme is by the microorganisms that belongs to bacillus, and this microorganism is bacillus unknown species SD114 (FERM BP-5736).
8. bacillus unknown species SD 114 (FERM BP-5736) and its mutant.
9. the composition of stain remover is comprising the proteolytic enzyme of claim 1-5 defined.
10. the stain remover that comprises the proteolytic enzyme of claim 1-5 defined.
11. be used for the stain remover of automatic dishwasher, wherein comprise the proteolytic enzyme of claim 1-5 defined.
12. produce peptide or amino acid whose method, it is characterized in that making proteolytic enzyme and the protein or the reactive polypeptide of each defined of claim 1-5.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP308493/95 | 1995-11-02 | ||
| JP308493/1995 | 1995-11-02 | ||
| JP30849395A JP4030603B2 (en) | 1995-11-02 | 1995-11-02 | Alkaline protease, method for producing the same, use and microorganism producing the protease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1201489A true CN1201489A (en) | 1998-12-09 |
| CN1125175C CN1125175C (en) | 2003-10-22 |
Family
ID=17981681
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96198013A Expired - Fee Related CN1125175C (en) | 1995-11-02 | 1996-11-01 | Alkaline protease, process for production thereof, using method thereof and microorganism producing same |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US5981255A (en) |
| EP (1) | EP0859050B1 (en) |
| JP (1) | JP4030603B2 (en) |
| CN (1) | CN1125175C (en) |
| AT (1) | ATE280222T1 (en) |
| AU (1) | AU7339596A (en) |
| DE (1) | DE69633676T2 (en) |
| WO (1) | WO1997016541A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303210C (en) * | 2000-12-29 | 2007-03-07 | 四川大学 | Detersile proteinase and its production process and usage and microbe producing the said proteinase |
| US7776579B2 (en) | 2002-10-24 | 2010-08-17 | Meiji Seika Kaisha, Ltd. | Method of degrading hardly degradable protein |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6180585B1 (en) | 1999-04-16 | 2001-01-30 | Spartan Chemical Company, Inc. | Aqueous disinfectant and hard surface cleaning composition and method of use |
| US6165965A (en) * | 1999-04-16 | 2000-12-26 | Spartan Chemical Company, Inc. | Aqueous disinfectant and hard surface cleaning composition and method of use |
| WO2000073429A1 (en) * | 1999-05-27 | 2000-12-07 | Amano Enzyme Inc. | Enzyme liquor and process for producing the same, enzyme preparation, protease preparations and protease-producing bacterium |
| US6387874B1 (en) | 2001-06-27 | 2002-05-14 | Spartan Chemical Company, Inc. | Cleaning composition containing an organic acid and a spore forming microbial composition |
| DE10163883A1 (en) * | 2001-12-22 | 2003-07-10 | Henkel Kgaa | New alkaline protease from Bacillus sp. (DSM 14390) and detergents and cleaning agents containing this new alkaline protease |
| WO2003062400A2 (en) * | 2002-01-24 | 2003-07-31 | The Scripps Research Institute | Fiber shaft modifications for efficient targeting |
| CN101389229B (en) * | 2006-01-04 | 2014-09-17 | 利品乐食品公司 | Protein hydrolysates and method of making |
| TWI434926B (en) * | 2006-12-11 | 2014-04-21 | Alcon Res Ltd | Use of peo-pbo block copolymers in ophthalmic compositions |
| TWI547522B (en) * | 2009-07-07 | 2016-09-01 | 愛爾康研究有限公司 | Ethyleneoxide butyleneoxide block copolymer compositions |
| WO2011153349A1 (en) | 2010-06-02 | 2011-12-08 | Alcon Research, Ltd. | Ophthalmic compositions comprising pbo-peo-pbo block copolymers |
| EP3486303A1 (en) * | 2017-11-21 | 2019-05-22 | Henkel IP & Holding GmbH | Laundry composition having broad spectrum stain removal |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5039151B2 (en) * | 1972-09-02 | 1975-12-15 | ||
| CA1186957A (en) * | 1982-06-07 | 1985-05-14 | Vincent Geracitano | Inflatable reversible liferaft |
| DK386586D0 (en) * | 1986-08-14 | 1986-08-14 | Novo Industri As | PROTEASE, PROCEDURE FOR PREPARING IT AND USING THEREOF |
| DK467387A (en) * | 1987-01-16 | 1988-07-17 | Goodrich Co B F | LIFEFLOW |
| JPH02255087A (en) * | 1989-03-27 | 1990-10-15 | Kurita Water Ind Ltd | Heat-resistant alkali protease and bacillus sp. no. ah-101 strain |
| DK58591D0 (en) * | 1991-04-03 | 1991-04-03 | Novo Nordisk As | HIS UNKNOWN PROTEAS |
| CA2066556A1 (en) * | 1991-04-26 | 1992-10-27 | Toyoji Sawayanagi | Alkaline protease, method for producing the same, use thereof and microorganism producing the same |
| US5275945A (en) * | 1991-10-08 | 1994-01-04 | Vista Chemical Company | Alkaline proteases stable in heavy-duty detergent liquids |
| EP0571014B1 (en) * | 1992-05-18 | 2004-03-31 | Genencor International, Inc. | Bacteria producing alkaline proteases, and production of these alkaline proteases |
| DE4218448A1 (en) * | 1992-06-04 | 1993-12-09 | Solvay Enzymes Gmbh & Co Kg | Alkaline proteases from Bacillus pumilus |
| JP2794371B2 (en) * | 1992-12-22 | 1998-09-03 | 花王株式会社 | Alkaline protease K-16H |
| US5397258A (en) * | 1993-12-30 | 1995-03-14 | Switlik Parachute Company, Inc. | Polygonally shaped inflatable raft apparatus |
| DE4411223A1 (en) * | 1994-03-31 | 1995-10-05 | Solvay Enzymes Gmbh & Co Kg | Use of alkaline proteases in commercial textile washing processes |
| JP3442475B2 (en) * | 1994-04-28 | 2003-09-02 | キョーラク株式会社 | Production method of filled tofu |
| JP2509540B2 (en) * | 1994-11-01 | 1996-06-19 | 花王株式会社 | Novel microorganism that produces new protease |
-
1995
- 1995-11-02 JP JP30849395A patent/JP4030603B2/en not_active Expired - Fee Related
-
1996
- 1996-11-01 DE DE69633676T patent/DE69633676T2/en not_active Expired - Lifetime
- 1996-11-01 EP EP96935529A patent/EP0859050B1/en not_active Expired - Lifetime
- 1996-11-01 AU AU73395/96A patent/AU7339596A/en not_active Abandoned
- 1996-11-01 US US09/043,722 patent/US5981255A/en not_active Expired - Lifetime
- 1996-11-01 AT AT96935529T patent/ATE280222T1/en not_active IP Right Cessation
- 1996-11-01 CN CN96198013A patent/CN1125175C/en not_active Expired - Fee Related
- 1996-11-01 WO PCT/JP1996/003216 patent/WO1997016541A1/en active IP Right Grant
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303210C (en) * | 2000-12-29 | 2007-03-07 | 四川大学 | Detersile proteinase and its production process and usage and microbe producing the said proteinase |
| US7776579B2 (en) | 2002-10-24 | 2010-08-17 | Meiji Seika Kaisha, Ltd. | Method of degrading hardly degradable protein |
Also Published As
| Publication number | Publication date |
|---|---|
| DE69633676T2 (en) | 2006-02-23 |
| WO1997016541A1 (en) | 1997-05-09 |
| EP0859050A4 (en) | 2001-10-17 |
| JPH09121857A (en) | 1997-05-13 |
| EP0859050B1 (en) | 2004-10-20 |
| AU7339596A (en) | 1997-05-22 |
| DE69633676D1 (en) | 2004-11-25 |
| EP0859050A1 (en) | 1998-08-19 |
| JP4030603B2 (en) | 2008-01-09 |
| US5981255A (en) | 1999-11-09 |
| CN1125175C (en) | 2003-10-22 |
| ATE280222T1 (en) | 2004-11-15 |
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